Establishment of a stable transfection method in Babesia microti and identification of a novel bidirectional promoter of Babesia microti

Abstract Babesia microti, an emerging human pathogen, is primarily transmitted through a bite of an infected tick and blood transfusions in human. Stable transfection technique has been reported in many protozoan parasites over the past few years. However, in vivo transient and stable transfection method has not been established for Babesia microti. Here, for the first time, we present a method of transient as well as stable transfection of the Babesia microti (B. microti) in the in vivo conditions. We have identified a novel promoter of B. microti. We also demonstrated that Plasmodium berghei DHFR promoter is recognized and functional in B. microti. We show that BM-CTQ41297 promoter control the expression of two genes, which are present on either side and thus represents a bi-functional promoter in B. microti. The predicted promoter activity values using Promoter 2.0 program is higher for BM- CTQ41297 promoter than strong promoters such as β-actin, ef-1β, and many other promoters. Furthermore, we discovered a non-essential locus for the genetic manipulation of the parasite, allowing us to stably integrate foreign genes; GFP, mCherry, into the B. microti. The transfection using an electroporation method and genetic manipulation of B. microti is now achievable and it is possible to obtain transfected viable parasites under in vivo growing conditions. The growth curve analysis of transfected and WT B. microti are similar indicating no defects in the transgenic parasites. This study will enable other researchers in understanding the B. microti biology, host modulation and diverse parasite developmental stages using reverse genetics and holds great potential to identify novel drug targets and vaccine development.

Download Full-text

HCV genome-wide analysis for development of efficient culture systems and unravelling of antiviral resistance in genotype 4

Gut ◽

10.1136/gutjnl-2020-323585 ◽

2021 ◽

pp. gutjnl-2020-323585

Author(s):

Long V. Pham ◽

Martin Schou Pedersen ◽

Ulrik Fahnøe ◽

Carlota Fernandez-Antunez ◽

Daryl Humes ◽

...

Keyword(s):

Drug Targets ◽

Reverse Genetics ◽

Complex Dynamics ◽

Antiviral Resistance ◽

Genotype 4 ◽

Culture Systems ◽

Direct Acting Antiviral ◽

Genome Wide ◽

Increased Risk

ObjectiveHCV-genotype 4 infections are a major cause of liver diseases in the Middle East/Africa with certain subtypes associated with increased risk of direct-acting antiviral (DAA) treatment failures. We aimed at developing infectious genotype 4 cell culture systems to understand the evolutionary genetic landscapes of antiviral resistance, which can help preserve the future efficacy of DAA-based therapy.DesignHCV recombinants were tested in liver-derived cells. Long-term coculture with DAAs served to induce antiviral-resistance phenotypes. Next-generation sequencing (NGS) of the entire HCV-coding sequence identified mutation networks. Resistance-associated substitutions (RAS) were studied using reverse-genetics.ResultThe in-vivo infectious ED43(4a) clone was adapted in Huh7.5 cells, using substitutions identified in ED43(Core-NS5A)/JFH1-chimeric viruses combined with selected NS5B-changes. NGS, and linkage analysis, permitted identification of multiple genetic branches emerging during culture adaptation, one of which had 31 substitutions leading to robust replication/propagation. Treatment of culture-adapted ED43 with nine clinically relevant protease-DAA, NS5A-DAA and NS5B-DAA led to complex dynamics of drug-target-specific RAS with coselection of genome-wide substitutions. Approved DAA combinations were efficient against the original virus, but not against variants with RAS in corresponding drug targets. However, retreatment with glecaprevir/pibrentasvir remained efficient against NS5A inhibitor and sofosbuvir resistant variants. Recombinants with specific RAS at NS3-156, NS5A-28, 30, 31 and 93 and NS5B-282 were viable, but NS3-A156M and NS5A-L30Δ (deletion) led to attenuated phenotypes.ConclusionRapidly emerging complex evolutionary landscapes of mutations define the persistence of HCV-RASs conferring resistance levels leading to treatment failure in genotype 4. The high barrier to resistance of glecaprevir/pibrentasvir could prevent persistence and propagation of antiviral resistance.

Download Full-text

Mammalian deubiquitinating enzyme inhibitors display in vitro and in vivo activity against malaria parasites and potentiate artemisinin action

10.1101/2020.08.13.249425 ◽

2020 ◽

Author(s):

Nelson V. Simwela ◽

Katie R. Hughes ◽

Michael T. Rennie ◽

Michael P. Barrett ◽

Andrew P. Waters

Keyword(s):

Anticancer Agents ◽

Drug Targets ◽

Reverse Genetics ◽

Enzyme Inhibitors ◽

Artemisinin Resistance ◽

Drug Repurposing ◽

Antimalarial Drugs ◽

Malaria Parasites

AbstractCurrent malaria control efforts rely significantly on artemisinin combinational therapies which have played massive roles in alleviating the global burden of the disease. Emergence of resistance to artemisinins is therefore, not just alarming but requires immediate intervention points such as development of new antimalarial drugs or improvement of the current drugs through adjuvant or combination therapies. Artemisinin resistance is primarily conferred by Kelch13 propeller mutations which are phenotypically characterised by generalised growth quiescence, altered haemoglobin trafficking and downstream enhanced activity of the parasite stress pathways through the ubiquitin proteasome system (UPS). Previous work on artemisinin resistance selection in a rodent model of malaria, which we and others have recently validated using reverse genetics, has also shown that mutations in deubiquitinating enzymes, DUBs (upstream UPS component) modulates susceptibility of malaria parasites to both artemisinin and chloroquine. The UPS or upstream protein trafficking pathways have, therefore, been proposed to be not just potential drug targets, but also possible intervention points to overcome artemisinin resistance. Here we report the activity of small molecule inhibitors targeting mammalian DUBs in malaria parasites. We show that generic DUB inhibitors can block intraerythrocytic development of malaria parasites in vitro and possess antiparasitic activity in vivo and can be used in combination with additive effect. We also show that inhibition of these upstream components of the UPS can potentiate the activity of artemisinin in vitro as well as in vivo to the extent that ART resistance can be overcome. Combinations of DUB inhibitors anticipated to target different DUB activities and downstream 20s proteasome inhibitors are even more effective at improving the potency of artemisinins than either inhibitors alone providing proof that targeting multiple UPS activities simultaneously could be an attractive approach to overcoming artemisinin resistance. These data further validate the parasite UPS as a target to both enhance artemisinin action and potentially overcome resistance. Lastly, we confirm that DUB inhibitors can be developed into in vivo antimalarial drugs with promise for activity against all of human malaria and could thus further exploit their current pursuit as anticancer agents in rapid drug repurposing programs.Graphical abstract

Download Full-text

Identification of a Novel Virulence Factor in Recombinant Pneumonia Virus of Mice

Journal of Virology ◽

10.1128/jvi.00364-07 ◽

2007 ◽

Vol 81 (17) ◽

pp. 9490-9501 ◽

Cited By ~ 18

Author(s):

Christine D. Krempl ◽

Anna Wnekowicz ◽

Elaine W. Lamirande ◽

Giw Nayebagha ◽

Peter L. Collins ◽

...

Keyword(s):

Virulence Factor ◽

Reverse Genetics ◽

Fluorescent Protein ◽

Vaccine Development ◽

Virulent Strain ◽

Consensus Sequence ◽

Cytoplasmic Tail ◽

Syncytial Virus

ABSTRACT Pneumonia virus of mice (PVM) is a murine relative of human respiratory syncytial virus (HRSV). Here we developed a reverse genetics system for PVM based on a consensus sequence for virulent strain 15. Recombinant PVM and a version engineered to express green fluorescent protein replicated as efficiently as the biological parent in vitro but were 4- and 12.5-fold attenuated in vivo, respectively. The G proteins of HRSV and PVM have been suggested to contribute to viral pathogenesis, but this had not been possible to study in a defined manner in a fully permissive host. As a first step, we evaluated recombinant mutants bearing a deletion of the entire G gene (ΔG) or expressing a G protein lacking its cytoplasmic tail (Gt). Both G mutants replicated as efficiently in vitro as their recombinant parent, but both were nonpathogenic in mice at doses that would otherwise be lethal. We could not detect replication of the ΔG mutant in mice, indicating that its attenuation is based on a severe reduction in the virus load. In contrast, the Gt mutant appeared to replicate as efficiently in mice as its recombinant parent. Thus, the reduction in virulence associated with the Gt mutant could not be accounted for by a reduction in viral replication. These results identified the cytoplasmic tail of G as a virulence factor whose effect is not mediated solely by the viral load. In addition to its intrinsic interest, a recombinant virus that replicates with wild-type-like efficiency but does not cause disease defines optimal properties for vaccine development.

Download Full-text

An improved method for the rescue of recombinant Newcastle disease virus

BioTechniques ◽

10.2144/btn-2019-0110 ◽

2020 ◽

Vol 68 (2) ◽

pp. 96-100

Author(s):

Pheik-Sheen Cheow ◽

Tiong Kit Tan ◽

Adelene Ai-Lian Song ◽

Khatijah Yusoff ◽

Suet Lin Chia

Keyword(s):

Newcastle Disease Virus ◽

Disease Virus ◽

Newcastle Disease ◽

Reverse Genetics ◽

Vaccine Development ◽

Transfection Efficiency ◽

Transfection Method ◽

Helper Plasmids ◽

Immunogenic Properties ◽

Recombinant Newcastle Disease Virus

Reverse genetics has been used to generate recombinant Newcastle disease virus with enhanced immunogenic properties for vaccine development. The system, which involves co-transfecting the viral antigenomic plasmid with three helper plasmids into a T7 RNA polymerase-expressing cell to produce viral progenies, poses a great challenge. We have modified the standard transfection method to improve the transfection efficiency of the plasmids, resulting in a higher titer of virus progeny production. Two transfection reagents (i.e., lipofectamine and polyethylenimine) were used to compare the transfection efficiency of the four plasmids. The virus progenies produced were quantitated with flow cytometry analysis of the infectious virus unit. The modified transfection method increased the titer of virus progenies compared with that of the standard transfection method.

Download Full-text

A Novel Approach To Display Structural Proteins of Hepatitis C Virus Quasispecies in Patients Reveals a Key Role of E2 HVR1 in Viral Evolution

Journal of Virology ◽

10.1128/jvi.00622-20 ◽

2020 ◽

Vol 94 (17) ◽

Author(s):

Yimin Tong ◽

Qingchao Li ◽

Rui Li ◽

Yongfen Xu ◽

Yu Pan ◽

...

Keyword(s):

Reverse Genetics ◽

Vaccine Development ◽

Viral Evolution ◽

Hcv Infection ◽

Clinical Isolates ◽

Structural Proteins ◽

Structural Protein ◽

Protein Coding ◽

Viral Structural Proteins

ABSTRACT Hepatitis C virus (HCV) infection remains a major worldwide health problem despite development of highly effective direct-acting antivirals. HCV rapidly evolves upon acute infection and generates multiple viral variants (quasispecies), leading to immune evasion and persistent viral infection. Identification of epitopes of broadly neutralizing anti-HCV antibodies (nAbs) is critical to guide HCV vaccine development. In this study, we developed a new reverse genetics system for HCV infection based on trans-complementation of viral structural proteins. The HCV genome (JFH1 strain) lacking the structural protein-coding sequence can be efficiently rescued by ectopic expression of core-E1-E2-p7-NS2 (core-NS2) or core-E1-E2-p7 (core-p7) in trans, leading to production of single-round infectious virions designated HCVΔS. JFH1-based HCVΔS can be also rescued by expressing core-NS2 of other HCV genotypes, rendering it an efficient tool to display the structural proteins of HCV strains of interests. Furthermore, we successfully rescued HCVΔS with structural proteins from clinical isolates. Multiple viral structural proteins with different sensitivities to nAbs were identified from a same patient serum, demonstrating the genetic diversity of HCV quasispecies in vivo. Interestingly, the structural protein-coding sequences of highly divergent viral quasispecies from the same patient can be clustered based on their hypervariable region 1 (HVR1) in viral envelope protein E2, which critically dictates the sensitivity to neutralizing antibodies. In summary, we developed a novel reverse genetics system that efficiently displays viral structural proteins from HCV clinical isolates, and analysis of quasispecies from the same patient using this system demonstrated that E2 HVR1 is the major determinant of viral evolution in vivo. IMPORTANCE A cell culture model that can recapitulate the diversity of HCV quasispecies in patients is important for analysis of neutralizing epitopes and HCV vaccine development. In this study, we developed a new reverse genetics system for HCV infection based on trans-complementation of viral structural proteins (HCVΔS). This system can be used to display structural proteins of HCV strains of multiple genotypes as well as clinical isolates. By using this system, we showed that multiple different HCV structural proteins from a same patient were displayed on HCVΔS. Interestingly, these variant structural proteins within the same patient can be classified according to the sequence of HVR1in E2, which dictates viral sensitivity to nAbs and viral evolution in vivo. Our work provided a new tool to study highly divergent HCV quasispecies and shed light on underlying mechanisms driving HCV evolution.

Download Full-text

Genetic Engineering of Treponema pallidum subsp. pallidum, the Syphilis Spirochete

10.1101/2021.05.07.443079 ◽

2021 ◽

Author(s):

Emily Romeis ◽

Lauren Tantalo ◽

Nicole Lieberman ◽

Quynh Phung ◽

Alex Greninger ◽

...

Keyword(s):

Genetic Engineering ◽

Vaccine Development ◽

Genetic Manipulation ◽

Treponema Pallidum ◽

Gene Promoter ◽

Digital Pcr ◽

Kanamycin Resistance ◽

In Vitro Cultivation

Background. Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum ( T. pallidum ), the causative of agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA ( tp0009 ) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance ( kan R ) cassette. Principal findings. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kan R gene was cloned downstream of the tp0574 gene promoter. The tp0574 prom- kan R cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kan R cassette into T. pallidum chromosome, in vitro -cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl 2 -based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kan R cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole genome sequencing (WGS) of transformed treponemes propagated in vitro and in vivo . ddPCR analysis of RNA and mass spectrometry confirmed expression of the kan R message and protein in treponemes propagated in vitro . Moreover, tprA knockout ( tprA ko -SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. Conclusion. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.

Download Full-text

Challenges in Drug Discovery against Tuberculosis

10.5772/intechopen.97857 ◽

2021 ◽

Author(s):

Manish Dwivedi ◽

Priya Giri

Keyword(s):

Drug Discovery ◽

Drug Targets ◽

Vaccine Development ◽

Extensive Literature ◽

Antitubercular Drugs ◽

Inadequate Information ◽

Adolescents And Adults ◽

Potent Drug ◽

Novel Drug

Tuberculosis (TB) is one of the deadly diseases in the present era caused by Mycobacterium tuberculosis. Principally, this bacterium attacks the lungs, however, MTB Has been observed affecting any part of the human body including the kidney, spine, and brain. Drug-resistant progression and other associated properties of MTB become a major hurdle in drug discovery to fight against tuberculosis. Moreover, some of the challenging situations such as the low range of chemical agents, the time-consuming process of drug development, the shortage of predictive animal models, and inadequate information of the physicochemical evidence required for effective bacterial penetration, are additional hindrances for the pharmaceutical scientist. In the current chapter, we focus on challenges encountered during drug discovery and need to be overcome as M. tuberculosis has a substantial barrier in its lipid-containing cell wall to inhibit the influx of drugs which is the initial requirement of the drug to show its therapeutic effect. There is also an immediate need for efficient vaccine development which may show its effect on adolescents and adults along with infants. Investigation on key bacterial targets has been troublesome, in light of the vulnerability around the microenvironments found in vivo and subsequently, the importance of exceptional metabolic pathways. The manuscript is prepared after the extensive literature survey to explore the vigorous approaches in novel drug designing and in proposing potent drug targets. The re-engineering and repositioning of prominent antitubercular drugs are required to attain viable control.

Download Full-text

Transient Transfection of the Zoonotic Parasite Babesia microti

Pathogens ◽

10.3390/pathogens9020108 ◽

2020 ◽

Vol 9 (2) ◽

pp. 108 ◽

Cited By ~ 1

Author(s):

Mingming Liu ◽

Shengwei Ji ◽

Mohamed Abdo Rizk ◽

Paul Franck Adjou Moumouni ◽

Eloiza May Galon ◽

...

Keyword(s):

Genetic Manipulation ◽

Firefly Luciferase ◽

Transient Transfection ◽

Babesia Microti ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Transfection Method ◽

Firefly Luciferase Reporter ◽

Zoonotic Parasite ◽

Firefly Luciferase Reporter Gene

The development of genetic manipulation techniques has been reported in many protozoan parasites over the past few years. However, these techniques have not been established for Babesia microti. Here, we report the first successful transient transfection of B. microti. The plasmids containing the firefly luciferase reporter gene were transfected into B. microti by an AMAXA 4D Nucleofection system. Twenty-four-hour synchronization, the 5′-actin promoter, program FA100, and 50 μg of plasmid DNA constituted the best conditions for the transient transfection of B. microti. This finding is the first step towards a stable transfection method for B. microti, which may contribute to a better understanding of the biology of the parasite.

Download Full-text

Stable transfection system for Babesia sp. Xinjiang

Parasites & Vectors ◽

10.1186/s13071-021-04940-x ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Jinming Wang ◽

Xiaoxing Wang ◽

Guiquan Guan ◽

Jifei Yang ◽

Junlong Liu ◽

...

Keyword(s):

Drug Targets ◽

Fluorescent Protein ◽

Genetic Manipulation ◽

Elongation Factor ◽

Pcr Amplification ◽

Basic Research ◽

Babesia Bovis ◽

Stable Transfection ◽

Drug Selection ◽

Babesia Gibsoni

Abstract Background Stable transfection systems have been described in many protozoan parasites, including Plasmodium falciparum, Cryptosporidium parvum, Babesia bovis, Babesia ovata, and Babesia gibsoni. For Babesia sp. Xinjiang (Bxj), which is the causative pathogen of ovine babesiosis and mainly prevails across China, the platform of those techniques remains absent. Genetic manipulation techniques are powerful tools to enhance our knowledge on parasite biology, which may provide potential drug targets and diagnostic markers. Methods We evaluated the inhibition efficiency of blasticidin (BSD) and WR99210 to Bxj. Then, a plasmid was constructed bearing selectable marker BSD, green fluorescent protein (GFP) gene, and rhoptry-associated protein-1 3′ terminator region (rap 3′ TR). The plasmid was integrated into the elongation factor-1 alpha (ef-1α) site of Bxj genome by cross-over homologous recombination technique. Twenty μg of plasmid was transfected into Bxj merozoites. Subsequently, drug selection was performed 24 h after transfection to generate transfected parasites. Results Transfected parasite lines, Bxj-c1, Bxj-c2, and Bxj-c3, were successfully obtained after transfection, drug selection, and colonization. Exogenous genes were integrated into the Bxj genome, which were confirmed by PCR amplification and sequencing. In addition, results of western blot (WB) and indirect immunofluorescence assay (IFA) revealed that GFP-BSD had expressed for 11 months. Conclusions In our present study, stable transfection system for Bxj was successfully developed. We anticipate that this platform will greatly facilitate basic research of Bxj. Graphical abstract

Download Full-text

Genetic engineering of Treponema pallidum subsp. pallidum, the Syphilis Spirochete

PLoS Pathogens ◽

10.1371/journal.ppat.1009612 ◽

2021 ◽

Vol 17 (7) ◽

pp. e1009612

Author(s):

Emily Romeis ◽

Lauren Tantalo ◽

Nicole Lieberman ◽

Quynh Phung ◽

Alex Greninger ◽

...

Keyword(s):

Genetic Engineering ◽

Vaccine Development ◽

Genetic Manipulation ◽

Treponema Pallidum ◽

Gene Promoter ◽

Digital Pcr ◽

Kanamycin Resistance ◽

Suicide Vector

Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kanR) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kanR gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kanR cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kanR cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kanR cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kanR message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.

Download Full-text